Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

Mihaela-Carmen Unciuleac,Yehuda Goldgur,Stewart Shuman

doi:10.1074/jbc.ra119.007445

Abstract

DNA ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA ligases join 3'-OH and 5'-PO4 ends via a series of three nucleotidyl transfer steps. In step 1, ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and release pyrophosphate (PPi) or nicotinamide mononucleotide. In step 2, AMP is transferred from ligase-adenylate to the 5'-PO4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA ligases are ATP-dependent. The catalytic core of ATP-dependent DNA ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4-1.8 Å resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (∼50 Å), from a closed conformation in the ATP complex to an open conformation in the covalent ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PPi leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg2+ that engages the ATP α phosphate and a second metal that bridges the ATP β and γ phosphates.

Highlights

DNA ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms
Many bacterial taxa (e.g. Mycobacterium, Pseudomonas, Agrobacterium) have a nonhomologous end-joining (NHEJ) system of DNA double-strand break (DSB) repair driven by Ku and one or two dedicated ATPdependent DNA ligases (LigD and LigC) [30]
LigD is a multifunctional enzyme composed of a ligase (LIG) domain fused to two other catalytic modules: a polymerase (POL) that preferentially adds ribonucleotides to DSB ends and a phosphoesterase that trims 3Ј oligoribo tracts until only a single 3Ј ribonucleotide remains

Summary

To whom correspondence should be addressed

Late or RNA-adenylate intermediate (AppDNA or AppRNA). In step 3, ligase catalyzes attack by a DNA or RNA 3Ј-OH on the polynucleotide-adenylate to seal the two ends via a phosphodiester bond and release AMP. Structures have been reported for T7 DNA ligase and human LigIV in complex with ATP [3, 13], but these are “off pathway” with respect to the lysine adenylylation reaction because the PPi leaving group (comprising the ATP ␤ and ␥ phosphates) is oriented orthogonal to the motif I lysine nucleophile (N␨–P␣–O3␣ angle of Յ90°), a situation inimical to an in line-attack by lysine on the ATP ␣ phosphate In these off-pathway ligase1⁄7ATP complexes, which lack a metal cofactor, the OB domain is splayed out away from the NTase domain, and there are no enzymic contacts to the ATP ␤ and ␥ phosphates. The structure of the MtuLigD-LIG domain, solved previously as the covalent ligase-(lysyl-N␨)–AMP intermediate [8], resembles other ATP-dependent DNA ligases with respect to its component NTase and OB domains and the conservation of its catalytic motifs. Enzymic contacts to the ATP ␥ phosphate orient the PPi leaving group favorably for in-line catalysis

Results

Discussion

Experimental procedures